Studies from this laboratory on the expression, assembly and topogenesis of intermediate filaments have indicated that they constitute an integral part of the structure and the morphogenetic program of certain differentiated lineages, notably muscle and erythroid cells. In chicken erythroid cells these filaments interlink the nucleus and the spectrin based membrane skeleton while in muscle cells they interlink myofibrils laterally to each other and to the membrane skeleton. Studies with cloned cDNA probes specific for the major filament subunits, desmin and vitamin, in these two cell lineages, as well as other related studies have established that a) expression of these two subunits is regulated at the mRNA level, b) expression of the two subunits is regulated independently in a lineage-specific fashion, c) the level of the polymer of each subunit is regulated primarily by the abundance of its mRNA and d) the ultimate cytoplasmic topogenesis of the filaments is regulated post-translationally. In chicken erythroid cells vitamin mRNA levels increase dramatically (about 50 fold) during embryo development. During this time the cells accumulate predominantly one vitamin transcript while otherwise in other cell types they accumulate two transcripts from a single gene. On the other hand, in mammalian erythroid development where the cells become enucleated, using murine erytholeukemia (MEL) cells as a model system, we have observed that vitamin mRNA levels decline precipitously after DMSO induced differentiation and intermediate filaments are removed. Here we propose experiments aimed at investigating the regulatory processes of the tissue specificity and developmental expression of desmin and vitamin. We plan to fully characterize the chicken vitamin and desmin genes which we have already isolated by conventional techniques. The difference in the two vitamin mRNA transcripts we have shown to reside in the 3' untranslated region of the gene generated by utilization of different polyadenylation sites and we plan to determine whether this difference, and their differential expression in erythroid cells, is due to differential termination of transcription or differential processing of the same transcriptional unit. These studies and other proposed studies aimed at investigating the metabolism of the two RNA species should define the regulation of vimentin accumulation in erythroid cells during chicken embryo development. Concurrently we plan transfection studies of the chicken vimentin gene into MEL cells, and upon induction of the cells to differentiate examine the mode of regulation of the transfected chicken gene in comparison to the endogenous mouse gene. Parallel studies with the desmin gene, a gene normally expressed only in muscle cells, transfected mammalian cells, should allow us a closer molecular understanding of the lineage specific expression of these subunits and how their involvement in cellular morphogenesis is regulated at the molecular level.
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